The long-term objective of the research proposal is to understand the regulation of the Ca2+-ATPase, and its role in the control of contractility in the heart. Phospholamban (PLB) is an integral membrane protein of cardiac muscle, and is the primary regulatory mechanism by which beta-adrenergic stimulation is transduced into a change in myocardial function. Through a presumed interaction with the Ca2+-pumping ATPase of cardiac sarcoplasmic reticulum (SR), phospholamban decreases the affinity of the Ca2+-ATPase for Ca2+. The physiological effect is thought to be altered Ca2+ loading of the SR which is linked to changes in contractility. Understanding this process at the molecular level will allow the design of therapeutic strategies targeting phospholamban in cardiac and smooth muscle, with potential benefits in sports medicine and the treatment of certain disease states (i.e., hypertrophy, congestive heart failure, hypertension, coronary artery disease, and asthma). Therefore, the specific aim of this research project is to characterize the structure of the inhibitory complex between PLB and Ca2+-ATPase. This will be achieved by co-reconstituting PLB and Ca2+-ATPase into a membrane environment at high density and crystallizing the resultant vesicles in the plane of the membrane. The structural interaction between these two proteins will be studied at an anticipated resolution of at least 14 angstroms by frozen hydrated electron microscopy and 3-dimensional helical reconstruction. In parallel, PLB will be reconstituted into a membrane environment at high density and crystallized in the plane of the membrane. The structure of this protein will be studied by frozen hydrated electron microscopy and 3-dimensional reconstruction. These structures will allow us to specifically define the site of PLB binding, the functional form of PLB, and conformational changes associated with PLB binding. Through this information, we will achieve a better understanding of the role of PLB in the regulation of contractility in cardiac muscle.